Zymogenic nucleic acid detection methods and related kits

ABSTRACT

This application provides methods of detecting and quantitatively determining a target nucleic acid sequence in a sample, which comprise contacting the sample with a primer and a zymogene which encodes, but which itself is the anti-sense sequence of, a catalytic nucleic acid sequence, so that when the target is present, a single amplified nucleic acid molecule is produced which comprises the sequences of both the target and catalytic molecules. This invention further provides a method of simultaneously detecting the presence of a plurality of target nucleic acid sequences in a sample. Finally, this invention provides molecules and kits for practicing the instant methods.

This application claims the benefit of U.S. provisional application No.60/076,899, filed Mar. 5, 1998.

Throughout this application, various publications are cited. Thedisclosure of these publications is hereby incorporated by referenceinto this application to describe more fully the state of the art towhich this invention pertains.

FIELD OF THE INVENTION

This invention relates to methods of detecting and quantitating targetnucleic acid molecules in a sample via nucleic acid amplification. Inthe instant methods, a single amplicon is produced containing bothcatalytic nucleic acid and target sequences. The catalytic nucleic acidis synthesized from its anti-sense, zymogenic precursor only if thetarget is present.

BACKGROUND OF THE INVENTION

Methods of in vitro nucleic acid amplification have wide-spreadapplications in genetics, disease diagnosis and forensics. In the lastdecade many techniques for amplification of known nucleic acid sequences("targets") have been described. These include the polymerase chainreaction ("PCR") (1-7, 41), the strand displacement amplification assay("SDA") (8) and transcription-mediated amplification ("TMA") (9, 10)(also known as self-sustained sequence replication ("SSR")). Theamplification products ("amplicons") produced by PCR and SDA are DNA,whereas RNA amplicons are produced by TMA. The DNA or RNA ampliconsgenerated by these methods can be used as markers of nucleic acidsequences associated with specific disorders.

Several methods allow simultaneous amplification and detection ofnucleic acids in a closed system, i.e., in a single homogeneous reactionsystem. These methods include Sunrise™ primers (11), Molecular Beacons(12) and the Taqman™ system (13). Using homogeneous sealed tube formatshas several advantages over separately analyzing amplicons followingamplification reactions. Closed system methods are faster and simplerbecause they require fewer manipulations. A closed system eliminates thepotential for false positives associated with contamination by ampliconsfrom other reactions. Homogeneous reactions can be monitored in realtime, with the signal at time zero allowing the measurement of thebackground signal in the system. Additional control reactions forestimating the background signal are therefore not required. A change inthe signal intensity indicates amplification of a specific nucleic acidsequence present in the sample.

Instead of amplifying the target nucleic acid, alternate strategiesinvolve amplifying the reporter signal. The Branched DNA assay (14)amplifies the signal by employing a secondary reporter molecule (e.g.alkaline phosphatase), whereas fluorescence correlation spectroscopy(FCS) employs electronic amplification of the signal (15).

As with other amplification technologies, catalytic nucleic acids havebeen studied intensively in recent years. The potential for suppressionof gene function using catalytic nucleic acids as therapeutic agents iswidely discussed in the literature (16-22). Catalytic RNA molecules("ribozymes") have been shown to catalyze the formation and cleavage ofphosphodiester bonds (16, 23). In vitro evolution techniques have beenused to discover additional nucleic acids which are capable ofcatalyzing a far broader range of reactions including cleavage (21, 22,24) and ligation of nucleic acids (25), porphyrin metallation (26), andformation of carbon-carbon (27), ester (28) and amide bonds (29).

Ribozymes have been shown to be capable of cleaving both RNA (16) andDNA (21) molecules. Similarly, catalytic DNA molecules ("DNAzymes") havealso been shown to be capable of cleaving both RNA (17, 24) and DNA (22,30) molecules. Catalytic nucleic acid can cleave a target nucleic acidsubstrate provided the substrate meets stringent sequence requirements.The target substrate must be complementary to the hybridizing regions ofthe catalytic nucleic acid and contain a specific sequence at the siteof cleavage. Examples of sequence requirements at the cleavage siteinclude the requirement for a purine:pyrimidine sequence for a class ofDNAzymes ("10-23 model" or "10-23 DNAzyme") (24), and the requirementfor the sequence U:X where X can equal A, C or U but not G, forhammerhead ribozymes (16).

In addition to having therapeutic potential, catalytic nucleic acidmolecules can also be used as molecular tools in genetic diagnosticassays. For example, ribozymes have been used to facilitate signalamplification in a two-stage method (31-33). In the first stage, a testsample is contacted with inactive oligonucleotides. This contactingresults in the production of "triggering" RNA oligonucleotides when thesample contains the target sequence. In the second stage the triggeringRNA oligonucleotides induce an amplification cascade. This cascaderesults in the production of large quantities of catalytically activereporter ribozymes which, when detected, indicate the presence of thetarget sequence in the test sample. The target sequence itself is notamplified during the process. Rather, only the reporter signal isamplified.

In short, target nucleic acid amplification and reaction conditionspermitting same are known. Catalytic nucleic acid molecules, andreaction conditions permitting their activity are also known.

However, no method has ever existed which permits the simultaneousprocesses of nucleic acid amplification and catalytic nucleic activityin a single reaction milieu. Moreover, no target amplification methodhas ever been performed wherein the amplification product is a singlenucleic acid molecule containing sequences for the target and thecatalytic nucleic acid molecule. Finally, no target amplification methodhas ever employed an anti-sense, zymogenic sequence of a catalyticnucleic acid molecule which, only in the presence of target sequence, isamplified in its "sense", catalytic form.

SUMMARY OF THE INVENTION

This invention provides a method of detecting the presence of a targetnucleic acid sequence in a sample which comprises

(a) contacting the sample, under conditions permitting primer-initiatednucleic acid amplification and catalytic nucleic acid activity, with

(i) a DNA primer suitable for initiating amplification of the target,and

(ii) a DNA zymogene which encodes, but which itself is the anti-sensesequence of, a catalytic nucleic acid molecule, wherein the primer andzymogene are situated with respect to each other so that, when thetarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and catalytic nucleicacid molecule; and

(b) determining the presence of catalytic nucleic acid activity, therebydetermining the presence of the target nucleic acid sequence in thesample.

This invention also provides a method of simultaneously detecting thepresence of a plurality of target nucleic acid sequences in a samplewhich comprises

(a) contacting the sample, under conditions permitting primer-initiatednucleic acid amplification and catalytic nucleic acid activity, with

(i) a plurality of primers wherein for each target being detected, thereexists at least one primer suitable for initiating amplification of thattarget, and

(ii) a plurality of zymogenes wherein for each target being detected,there exists at least one zymogene which encodes, but which itself isthe anti-sense sequence of, a catalytic nucleic acid molecule havingdistinctly measurable activity, the primer and zymogene being situatedwith respect to each other so that, when the corresponding target ispresent, a single amplified nucleic acid molecule is produced whichcomprises the sequences of both the target and corresponding catalyticnucleic acid molecule; and

(b) simultaneously determining the presence of each of the catalyticnucleic acid activities, thereby determining the presence of each of thecorresponding target nucleic acid sequences in the sample.

This invention further provides a DNA molecule comprising a primer and azymogene, wherein the primer is situated 3' of the zymogene.

This invention still further provides a kit for use in determining thepresence of a target nucleic acid sequence in a sample, which comprises

(a) a primer suitable for initiating amplification of the target;

(b) a zymogene which encodes, but which itself is the anti-sensesequence of, a catalytic nucleic acid sequence, wherein the primer andzymogene are situated with respect to each other so that, when thetarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and catalytic nucleicacid molecule; and

(c) reagents permitting primer-initiated nucleic acid amplification andcatalytic nucleic acid activity.

Finally, this invention provides a kit for use in determining thepresence of a plurality of target nucleic acid sequences in a sample,which comprises

(a) a plurality of primers, wherein for each target being detected,there exists at least one primer suitable for initiating amplificationof that target;

(b) a plurality of zymogenes wherein for each target being detected,there exists at least one zymogene which encodes, but which itself isthe anti-sense sequence of, a catalytic nucleic acid sequence havingdistinctly measurable activity, wherein the primer and zymogene aresituated with respect to each other so that, when the correspondingtarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and correspondingcatalytic nucleic acid molecule; and

(c) reagents permitting primer-initiated nucleic acid amplification andcatalytic nucleic acid activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of the instant method. Here, a nucleic acidmolecule comprising a primer and zymogene is contacted with a segment ofgenomic DNA comprising a target sequence. This gives rise to a secondnucleic acid molecule comprising a catalytic nucleic acid and a target.The catalytic nucleic acid in turn cleaves a detectable substrate.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In this invention, certain terms are used frequently which shall havethe meanings set forth as follows. "Catalytic nucleic acid molecule","catalytic nucleic acid", and "catalytic nucleic acid sequence" areequivalent, and each shall mean a DNA molecule or DNA-containingmolecule (also known in the art as a "DNAzyme") or an RNA orRNA-containing molecule (also known in the art as a "ribozyme") whichspecifically recognizes a distinct substrate and catalyzes the chemicalmodification of this substrate. The nucleic acid bases in the DNAzymesand ribozymes can be the bases A, C, G, T and U, as well as derivativesthereof. Derivatives of these bases are well known in the art, and areexemplified in reference 40.

"Amplification" of a target nucleic acid sequence shall mean theexponential amplification thereof (as opposed to linear amplification),whereby each amplification cycle doubles the number of target ampliconspresent immediately preceding the cycle. Methods of exponentialamplification include, but are not limited to, PCR, SDA and TMA.Exponential amplification differs from linear amplification, whereby inlinear amplification, each amplification cycle increases by a fixednumber the number of target amplicons present immediately preceding thecycle.

"Reporter substrate", "chemical substrate" and "substrate" areequivalent, and each shall mean any molecule which is specificallyrecognized and modified by a catalytic nucleic acid molecule. "Target"and "target nucleic acid sequence" are equivalent, and each shall meanthe nucleic acid sequence of interest to be detected or measured by theinstant invention, which comprises a sequence that hybridizes with theprimer when contacted therewith in this method, and that can be eitheran entire molecule or a portion thereof. "Primer" shall mean a shortsegment of DNA or DNA-containing nucleic acid molecule, which (i)anneals under amplification conditions to a suitable portion of a DNA orRNA sequence to be amplified, and (ii) initiates, and is itselfphysically extended, via polymerase-mediated synthesis. Finally,"zymogene" shall mean a nucleic acid sequence which comprises theanti-sense (i.e. complementary) sequence of a catalytic nucleic acidmolecule having detectable activity, and whose transcription product isthe catalytic nucleic acid molecule.

Embodiments of the Invention

This invention provides a rapid and procedurally flexible method ofdetecting and quantitatively measuring target nucleic acid sequences ofinterest in a sample. This method is unique in that it simultaneouslyemploys target amplification and detection via catalytic nucleicactivity in a single reaction vessel. Moreover, it is unique in that theamplification product is a single nucleic acid molecule containingsequences for the target and the catalytic nucleic acid molecule.Finally, this method is the first to employ an anti-sense, zymogenesequence of a catalytic nucleic acid molecule which--only in thepresence of the target sequence--is amplified in its "sense", catalyticform.

More specifically, this invention provides a method of detecting thepresence of a target nucleic acid sequence in a sample which comprises

(a) contacting the sample, under conditions permitting primer-initiatednucleic acid amplification and catalytic nucleic acid activity, with

(i) a DNA primer suitable for initiating amplification of the target,and

(ii) a DNA zymogene which encodes, but which itself is the anti-sensesequence of, a catalytic nucleic acid molecule, wherein the primer andzymogene are situated with respect to each other so that, when thetarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and catalytic nucleicacid molecule; and

(b) determining the presence of catalytic nucleic acid activity, therebydetermining the presence of the target nucleic acid sequence in thesample.

In one embodiment, the instant method further comprises the step ofquantitatively determining the amount of catalytic nucleic acid activityin the sample resulting from step (a), and comparing the amount ofactivity so determined to a known standard, thereby quantitativelydetermining the amount of the target nucleic acid sequence. The knownstandard can be any standard or control used for quantitativedetermination. Examples of these standards include (i) known reactionkinetic information, as well as (ii) signal measurements obtained usingsamples containing no catalytic activity, or a pre-determined amount ofcatalytic activity.

In one embodiment, the primer and zymogene are on separate DNAmolecules, and the primer-initiated nucleic acid amplification isrolling circle amplification (a known amplification method). In aanother embodiment, at least two of the DNA molecules comprise both theprimer and zymogene.

In a further embodiment, the sample is contacted with two DNA molecules,each molecule comprising a primer, and at least one molecule comprisingthe zymogene wherein the primer is situated 3' of the zymogene.

In one form of this embodiment, the DNAzyme encoded by the zymogenerecognizes and cleaves a sequence actually residing on the amplifiednucleic acid molecule itself (i.e., cis cleavage, as opposed to transcleavage whereby the DNAzyme cleaves a substrate located on a differentmolecule). More specifically, the single amplified nucleic acid moleculefurther comprises a nucleotide sequence recognized and cleaved in cis bythe zymogene-encoded catalytic nucleic acid DNAzyme co-residing on theamplified molecule.

In the preferred embodiment of this method employing cisDNAzyme-catalyzed cleavage, the zymogene-encoded DNAzyme is a 10-23DNAzyme, and the DNA primer used in step (a)(i) of the instant method(i.e., a "chimeric" primer) contains at least one purine ribonucleotideresidue which serves as the 5' side of the site recognized and cleavedin cis by the 10-23 DNAzyme. This purine ribonucleotide residue in thechimeric primer is required for cleavage by the 10-23 DNAzyme. Thus,using this chimeric primer permits the 10-23 DNAzyme cleavage site to begenerated in a PCR reaction. The chimeric primer can also include, forexample, a ribonucleotide residue that serves as the 3' side of the siterecognized and cleaved in cis by the 10-23 DNAzyme.

In this invention, the nucleic acid molecules comprising the primersand/or zymogenes can also comprise additional sequences, such assequences complementary to the target.

The target sequence detected or quantitated in the instant methods canbe any nucleic acid sequence. In one embodiment, the target nucleic acidsequence is a DNA molecule. In another embodiment, the target nucleicacid sequence is an RNA molecule, and step (a) further comprises therequired step of first reverse transcribing the target RNA sequence toDNA prior to contacting the sample with the primer and zymogene.

The catalytic nucleic acid molecule encoded by the zymogene can be aribozyme or a DNAzyme. In one embodiment, the catalytic nucleic acidmolecule is a ribozyme. In another embodiment, the catalytic nucleicacid molecule is a DNAzyme.

The catalytic nucleic acid activity measured in the instant methods canbe any activity which can occur (and, optionally, be measured)simultaneously and in the same milieu with a nucleic acid amplificationreaction. The catalytic nucleic acid activity can comprise, for example,the modification of a detectable chemical substrate, which modificationis selected from the group consisting of phosphodiester bond formationand cleavage, nucleic acid ligation and cleavage, porphyrin metallation,and formation of carbon-carbon, ester and amide bonds. In oneembodiment, the detectable chemical substrate modification is cleavageof a fluorescently labeled nucleic acid molecule, preferably a DNA/RNAchimera.

In the preferred embodiment, the reporter substrate is cleaved, andmeasuring this cleavage is a means of measuring the catalytic activity.For example, the presence of the cleaved substrate can be monitored byphosphorimaging following gel electrophoresis provided the reportersubstrate is radiolabelled. The presence of cleaved substrate can alsobe monitored by changes in fluorescence resulting from the separation offluoro/quencher dye molecules incorporated into opposite sides of thecleavage site within the substrate. Such systems provide the opportunityfor a homogeneous assay which can be monitored in real time. Methods formonitoring changes in fluorescence are well known in the art. Suchmethods include, by way of example, visual observation and monitoringwith a spectrofluorometer.

The target nucleic acid sequence can be from any organism, and thesample can be any composition containing, or suspected to contain,nucleic acid molecules. In one embodiment, the target is from a plant,or from an animal such as, for example, a mouse, rat, dog, guinea pig,ferret, rabbit, and primate. In another embodiment, the target is in asample obtained from a source such as water or soil. In a furtherembodiment, the target is from a sample containing bacteria, viruses ormycoplasma.

In the preferred embodiment, the target is from a human. The instantmethods can be used for a variety of purposes including, for example,diagnostic, public health and forensic.

In one embodiment, the instant method is used for diagnostic purposes.Specifically, the invention can be used to diagnose a disorder in asubject characterized by the presence of at least one target nucleicacid sequence which is not present when such disorder is absent. Suchdisorders are well known in the art and include, by way of example,cancer, cystic fibrosis, and various hemoglobinopathies. The inventioncan also be used to diagnose disorders associated with the presence ofinfectious agents. Such disorders include, by way of example, AIDS,Hepatitis C, and tuberculosis. In the preferred embodiment, the subjectbeing diagnosed is human and the disorder is cancer.

In another embodiment, the sample being tested for the presence oramount of target nucleic acid molecule is a sample taken for publichealth purposes. Examples of such samples include water, food and soil,possibly containing harmful pathogens such as bacteria, viruses andmycoplasma.

In a further embodiment, the sample being tested for the presence oramount of target nucleic acid molecules is a forensic sample. Examplesof such samples include bodily fluids, tissues and cells, which can beobtained from any source such as a crime scene.

This invention also provides a method of simultaneously detecting thepresence of a plurality of target nucleic acid sequences in a samplewhich comprises

(a) contacting the sample, under conditions permitting primer-initiatednucleic acid amplification and catalytic nucleic acid activity, with

(i) a plurality of primers wherein for each target being detected, thereexists at least one primer suitable for initiating amplification of thattarget, and

(ii) a plurality of zymogenes wherein for each target being detected,there exists at least one zymogene which encodes, but which itself isthe anti-sense sequence of, a catalytic nucleic acid molecule havingdistinctly measurable activity, the primer and zymogene being situatedwith respect to each other so that, when the corresponding target ispresent, a single amplified nucleic acid molecule is produced whichcomprises the sequences of both the target and corresponding catalyticnucleic acid molecule; and

(b) simultaneously determining the presence of each of the catalyticnucleic acid activities, thereby determining the presence of each of thecorresponding target nucleic acid sequences in the sample.

In one embodiment, the method of simultaneously detecting the presenceof a plurality of targets further comprising the step of quantitativelydetermining the amount of each catalytic nucleic acid activity in thesample resulting from step (a), and comparing the amount of eachactivity so determined to a known standard, thereby quantitativelydetermining the amount of each target nucleic acid sequence. Examples ofmultiple targets which can be simultaneously detected by the instantmethods are disclosed in reference 39.

This invention further provides a DNA molecule comprising a primer and azymogene, wherein the primer is situated 3' of the zymogene. The instantmolecule can be used pursuant to the instant methods.

This invention still further provides a kit for use in determining thepresence of a target nucleic acid sequence in a sample, which comprises

(a) a primer suitable for initiating amplification of the target;

(b) a zymogene which encodes, but which itself is the anti-sensesequence of, a catalytic nucleic acid sequence, wherein the primer andzymogene are situated with respect to each other so that, when thetarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and catalytic nucleicacid molecule; and

(c) reagents permitting primer-initiated nucleic acid amplification andcatalytic nucleic acid activity.

Finally, this invention provides a kit for use in determining thepresence of a plurality of target nucleic acid sequences in a sample,which comprises

(a) a plurality of primers, wherein for each target being detected,there exists at least one primer suitable for initiating amplificationof that target;

(b) a plurality of zymogenes wherein for each target being detected,there exists at least one zymogene which encodes, but which itself isthe anti-sense sequence of, a catalytic nucleic acid sequence havingdistinctly measurable activity, wherein the primer and zymogene aresituated with respect to each other so that, when the correspondingtarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and correspondingcatalytic nucleic acid molecule; and

(c) reagents permitting primer-initiated nucleic acid amplification andcatalytic nucleic acid activity.

In one embodiment, the instant kit further comprises reagents useful forisolating a sample of nucleic acid molecules from a subject or sample.The components in the instant kit can either be obtained commercially ormade according to well known methods in the art, as exemplified in theExperimental Details section below. In addition, the components of theinstant kit can be in solution or lyophilized as appropriate. In oneembodiment, the components are in the same compartment, and in anotherembodiment, the components are in separate compartments. In thepreferred embodiment, the kit further comprises instructions for use.

In the instant methods and kits, the nucleic acid amplification can beperformed according to any suitable method known in the art, andpreferably according to one selected from the group consisting of PCR,SDA and TMA.

Numerous methods are relevant to this invention which are within routineskill in the art. These include: methods for isolating nucleic acidmolecules, including, for example, phenol chloroform extraction, quicklysis and capture on columns (34-38); methods of detecting andquantitating nucleic acid molecules; methods of detecting andquantitating catalytic nucleic acid activity; methods of amplifying anucleic acid sequence including, for example, PCR, SDA and TMA (alsoknown as (SSR))(1-10, 41); methods of designing and making primers foramplifying a particular target sequence; and methods of determiningwhether a catalytic nucleic acid molecule cleaves an amplified nucleicacid segment including, by way of example, polyacrylamide gelelectrophoresis and fluorescence resonance energy transfer (FRET) (25,31).

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS Example 1 Detection of K-ras in Tumor Cell DNA

A. PCR Primers

Three PCR primers (5KID, 3K2Dz3 and 3K2) were synthesized by OligosEtc., Inc. (Wilsonville, Oreg., USA). The 5' PCR primer (5KID) iscomplementary to the human K-ras gene. The 3' primer 3K2Dz3 is azymogene PCR primer which contains (a) a 5' region containing thecatalytically inactive antisense sequence complementary to an activeDNAzyme and (b) a 3' region which is complementary to the human K-rasgene. During PCR amplification using 5KID and 3K2Dz3, the ampliconsproduced by extension of 5KID contain both K-ras sequences andcatalytically active sense copies of a DNAzyme incorporated in their 3'regions. The active DNAzyme is designed to cleave an RNA/DNA reportersubstrate (Sub 1). The primer 3K2 is a control primer which contains thesame K-ras-specific sequences which are incorporated in 3K2Dz3. Howeverthis primer contains no zymogene sequence. The sequences of the PCRprimers are listed below. Sequences underlined are complementary to thehuman K-ras gene and the sequence in bold is the inactive (antisense)sequence which is complementary to an active DNAzyme.

5KID(SEQ ID NO:1) (5' K-ras primer)

GGCCTGCTGAAAATGACTGAATA

3K2Dz3(SEQ ID NO:2) (3' K-ras zymogene primer)

GAGAACTGCAATTCGTTGTAGCTAGCCTTTCAGGACCCACGTCCA CAAAATGATTCTGA

3K2(SEQ ID NO:3) (3' K-ras primer for control reaction)

CGTCCACAAAATGATTCTGA

B. Reporter Substrate

The reporter substrate (Sub 1) was synthesized by Oligos Etc., Inc.(Wilsonville, Oreg., USA). Sub 1 is a chimeric molecule containing bothRNA (bold, underlined) and DNA bases. It has a 3' phosphate group whichprevents its extension by DNA polymerase during PCR. Sub 1 was 5'end-labeled with ³² P by standard techniques (34). The sequence of Sub 1is illustrated below.

Sub 1(SEQ ID NO:4)

GAGAACTGCAAUGUUTCAGGACCCA

C. DNAzyme for a Control Cleavage Reaction

The DNAzyme Dz3a was synthesized by Oligos Etc., Inc. (Wilsonville,Oreg., USA). This DNAzyme is designed to cleave Sub 1 at the samesequence which is cleaved by the active DNAzyme generated during PCRamplification using the zymogene primer 3K2Dz3. The sequence of Dz3a isillustrated below.

Dz3a (SEQ ID NO:5)

TCCTGAAAGGCTAGCTACAACGAATTGCAGT

D. Preparation of Genomic DNA from a Tumor Cell Line

The human cell line K562 was obtained from the American Type CultureCollection (Rockville, Md.). K562 is a leukemic cell line which harboursa wild type K-ras sequence. Genomic DNA was prepared by cationic polymerextraction (38).

E. PCR Amplification of the K-ras Gene

Genomic DNA isolated from K562 was amplified by PCR. Reactions (A1 andA2) contained genomic DNA (500 ng), 50 pmole of 5KID, 1 pmole of 3K2Dz3,50 fmole of ³² P-labeled Sub 1, each dNTP (dATP, dCTP, dTTP, dGTP) at100 uM in 100 mM NaCl, 50 mM Tris (pH 8.3 at 25° C.) and 8 mM MgCl₂. Sixunits of Taq DNA polymerase (5 units/ul; AmpliTaq, Perkin Elmer) weremixed with 2 ul of TaqStart™ antibody (1.1 mg/ml, Clontech) in 1.8 ul ofantibody dilution buffer (Clontech). The Taq DNA polymerase:TaqStart™antibody mixture was incubated for 15 minutes at room temperature priorto addition to the PCR mixture. The total reaction volumes were 50 ul.One negative control reaction (B) contained all reaction components withthe exception of genomic DNA. A second negative control reaction (C)contained all reaction components with the exception of 3K2Dz3 which wasreplaced with 1 pmole of 3K2. A positive control cleavage reaction (D)contained 30 pmole of Dz3a plus all reaction components present incontrol reaction C. The reactions were placed in a GeneAmp PCR system9600 (Perkin Elmer), denatured at 94° C. for 2 minutes and thensubjected to 20 cycles of 92° C. for 20 seconds and 58° C. for 30seconds, followed by 20 cycles of 92° C. for 20 seconds, 74° C. for 1second and 40° C. for 20 seconds.

F. Detection of Cleaved Reporter Substrate Sub 1

A 3 ul aliquot of each reaction was analyzed without subsequentmanipulation by electrophoresis on a 16% denaturing polyacrylamide gel.The gel was visualized by phosphorimagery on a PhosphoImager: 445 S1(Molecular Dynamics). The radiolabelled 25-base Sub 1 reporter substratewas cleaved to produce a radiolabelled fragment of 13 bases in thepositive control cleavage reaction D. The same 13-base fragment waspresent in the PCR reactions A1 and A2 which contained both genomic DNAand the zymogene primer 3K2Dz3, indicating successful amplification ofthe K-ras gene by PCR. In the negative control reaction B (whichcontained no genomic DNA), only the 25-base fragment was evident,indicating cleavage of the substrate does not occur in the absence ofamplification of target DNA. Finally, in the negative control reactionC, where the zymogene primer was replaced with a primer containing onlyK-ras sequences, the substrate was not cleaved since active DNAzymes arenot produced in this reaction.

Example 2 Cleavage of a Fluorescent Reporter Substrate

A. Reporter Substrate

The reporter substrate, SubCz2, was synthesized by Oligos Etc., Inc.(Wilsonville, Oreg., USA). The sequence of SubCz2 is illustrated below.SubCz2 is a chimeric molecule containing both RNA (shown below in lowercase) and DNA nucleotides. It has a 3' phosphate group that prevents itsextension by DNA polymerase during PCR. SubCz2 was synthesized with a6-carboxyfluorescein ("6-FAM") moiety attached to the 5' nucleotide(bold, underlined) and an N,N,N',N'-tetramethyl-6-carboxyrhodamine("TAMRA") moiety attached to the first "T" deoxyribonucleotide (bold,underlined) 3' to the RNA bases. The cleavage of the reporter substratecan be monitored at 530 nm (FAM emission wavelength) with excitation at485 nm (FAM excitation wavelength).

SubCz2(SEQ ID NO:6)

5' CCACTCguATTAGCTGTATCGTCAAGCCACTC 3'

B. PCR Primers

Two PCR primers were synthesised by Bresatec Pty. Ltd. (Adelaide, SA,Australia) or Pacific Oligos Pty. Ltd. (Lismore, NSW, Australia). The 5'PCR primer (5K49) is complementary to the human K-ras gene. The 3'primer (3K45Zc2) is a zymogene PCR primer which contains (a) a 5' regioncontaining the catalytically inactive antisense sequence of an activeDNAzyme and (b) a 3' region which is complementary to the human K-rasgene. During PCR amplification using 5K49 and 3K45Zc2, the ampliconsproduced by extension of 5K49 contain both K-ras sequences andcatalytically active sense copies of a DNAzyme incorporated in their 3'regions. The active DNAzyme is designed to cleave the RNA/DNA reportersubstrate SubCz2. The sequences of the PCR primers are illustratedbelow. The underlined portion of the sequence is complementary to thehuman K-ras gene, and the sequence shown in bold is the inactive(antisense) sequence that is complementary to the active DNAzyme.

5K49(SEQ ID NO:7) (5' K-ras primer)

5' CCTGCTGAAAATGACTGAATATAAA 3'

3K45Zc2(SEQ ID NO:8) (3' K-ras zymogene primer)

5' CCACTCTCGTTGTAGCTAGCCT

ATTAGCTGTATCGTCAAGCCACTCTTGC 3'

C. Preparation of Genomic DNA from a Tumor Cell Line

The human cell line K562 was obtained from the American Type CultureCollection (Rockville, Md.). K562 is a leukemic cell line that harboursa wild type K-ras sequence. Genomic DNA was prepared by cationic polymerextraction (38).

D. PCR Amplification of the K-ras Gene

Genomic DNA isolated from K562 was amplified by PCR. Reactions contained20 pmole 5K49, 3 pmole 3K45Zc2, 10 pmol SubCz2, 8 mM MgCl₂, 100 uM ofeach of dATP, dCTP, dTTP, and dGTP, and 1×buffer (75 mM KCl with 10 mMTris pH 8.3 at 25° C.). All solutions used in the PCR were made up inDEPC-treated water. Three units of Taq DNA polymerase (5 units/ulAmpliTaq, Perkin-Elmer) were mixed with TaqStart™ antibody (Clontech) togive a final molar ratio of Taq DNA polymerase:TaqStart™ antibody of1:10. The Taq DNA polymerase:TaqStart™ antibody mixture was incubatedfor 15 minutes at room temperature prior to addition to the PCR mixture.The total reaction volumes were made up to 50 ul. Duplicate reactionswere set up which contained 500 ng of K562 genomic DNA. Controlreactions contained all reaction components with the exception ofgenomic DNA. The reactions were placed in an ABI Prism 7700 SequenceDetection System and incubated at 40° C. for 1 minute (to provide a baseline reading), denatured at 94° C. for 3 minutes, subjected to 20 cyclesof 70° C. for 1 minute with a temperature decrease of 1° C. per cycle,and followed by incubation at 94° C. for 5 seconds. This was followed bya further 50 cycles at 40° C. for 1 minute, followed by incubation at94° C. for 5 seconds.

Fluorescence was measured by the ABI Prism 7700 Sequence DetectionSystem during the annealing/extension phase of the PCR. Reactions withgenomic DNA showed an increase in FAM fluorescence at 530 nm over thefluorescence observed in control reactions. This fluorescence increasewas used to monitor the accumulation of K-ras amplicons during PCR.These results confirm that zymogene PCR can be used to facilitatehomogeneous amplification and real time detection in a simplefluorescent format.

Example 3 Use of Zymogenes to Distinguish Between Variant Alleles:Detection of Mutations at K-ras Codon 12

A. Strategy

PCR using zymogene primers can also be used for the analysis of pointmutations. In this example, the zymogene primers facilitate synthesis ofactive DNAzymes during PCR. These DNAzymes are designed to cleave thePCR amplicons in cis only when their hybridizing arms are fullycomplementary to position 1 of codon 12 within K-ras. Walder, et al.(41) have previously shown that Taq DNA polymerase can extend DNA/RNAchimeric primers that contain one or two 3' terminal ribose residues.These chimeric primers are used here to produce PCR amplicons that serveas substrates for the 10-23 DNAzyme.

PCR using a 5' DNA/RNA chimeric primer (5K42r) and a 3' zymogene primer(3K42Dz2) amplified a region of K-ras. 5K42r hybridized to the K-rassequence adjacent to codon 12 and contained the purine:pyrimidineresidues which form the potential DNAzyme cleavage site. The zymogeneprimer 3K42Dz2 has a 3' region that is complementary to K-ras, and a 5'region that contains the antisense of a DNAzyme. The zymogene primer hadno inherent catalytic activity itself but, when used in conjunction with5K42r, it facilitated the production of amplicons having (a) DNAzymecleavage sites near their 5' termini and (b) active (sense) DNAzymes attheir 3' termini. This DNAzyme is designed to cleave the 5' end of theamplicons in cis. The 5' arm of the DNAzyme is fully complementary tosequences that are wild type at codon 12. Mutations at K-ras codon 12,which result in mismatches with the 5' DNAzyme arm, are predicted tosignificantly decrease the efficiency of DNAzyme cleavage.

B. Primer Sequences

5' chimeric primer 5K42r (SEQ ID NO:9)

(upper case--deoxyribonucleotide residues; lower case ribonucleotideresidues)

5' TATAAACTTGTGGTAGTTGGAgcT 3'

3' zymogene primer 3K42Dz2(SEQ ID NO:10)

(complement of 10:23 catalytic core in bold)

5' ACTTGTGGTAGTTGGATCGTTGTAGCTAGCCCTGG

TGGCAGCTGTATCGTCAAGGCACTC 3'

The primers were synthesised by Pacific Oligos Pty. Ltd. (Lismore, NSW,Australia) or Oligos Etc., Inc. (Wilsonville, Oreg., USA). The 5'primer, 5K42r, was 5' end-labelled with g-³² P by incubating 25 ul of 20uM primer with 2.5 ul of polynucleotide kinase (10×10³ U/ml, 3'phosphatase-free, Boehringer Mannheim), 2.5 ul rRNasin (40 U/ulrecombinant rRNasin®, ribonuclease inhibitor, Promega), 5 ul ofpolynucleotide kinase buffer (Boehringer Mannheim), 10 ul of g-³² PAdenosine 5'-triphosphate (2.5 uM, Stable Label Gold™, Bresatec) and 5ul of DEPC water for 30 minutes at 37° C.

C. K-ras DNA Templates

pUC 18 plasmid vectors containing K-ras exon 1 sequences, which wereeither wild type (GGT) or mutated at codon 12 (CGT or AGT), were used asDNA templates for PCR.

D. Amplification by Zymogene PCR and Cleavage by DNAzymes SynthesisedDuring the Reaction

PCR mixtures contained 0.2 pg/ul K-ras plasmid DNA, 10 pmole of g-³²P-labelled 5K42r, 2 pmole 3K42Dz2, 1 mM DTT, 8 mM MgCl₂, each dNTP(dATP, dCTP, dTTP, dGTP) at 100 uM, 0.4 U/ul rRNasin®, and 1×buffer (100mM NaCl with 50 mM Tris pH 8.3 at 25° C.). Duplicate reactions were setup for each DNA template. Six units of Taq DNA polymerase (5 units/ulAmpliTaq, Perkin-Elmer) were mixed with TaqStart™ antibody (Clontech) togive a final molar ratio of Taq DNA polymerase: TaqStart™ antibody of1:5. The Taq DNA polymerase: TaqStart™ antibody mixture was incubatedfor 15 minutes at room temperature prior to addition to the PCR mix. Thetotal reaction volumes were 50 ul. The reactions were placed in aGeneAmp PCR 9600 (Perkin-Elmer), denatured at 94° C. for 2 minutes,subjected to 30 cycles at 60° C. for 1 minute, followed by treatment at94° C. for 20 seconds. The reaction was further subjected to 10 cyclesat 50° C. for 1 minute, followed by treatment at 94° C. for 20 seconds.

A 2.5 ul aliquot of each reaction was mixed with 2.5 ul of loading dye(97.5% formamide, 0.1% xylene cyanol, 0.1% bromopheol blue and 0.01 MEDTA), incubated at 75° C. for 2 minutes, and then loaded immediatelyonto a pre-warmed 16% denaturing (urea) acrylamide gel. The gels wereelectrophoresed for approximately 1 hour. The PCR product and cleavagefragments were visualised by scanning the gel using a Molecular DynamicsPhosphorimager 445 S1.

Several bands were visible on the gel (data not shown). The fragments,in order of mobility from the slowest to the fastest (i.e., from theorigin to the bottom of the gel) were (a) PCR amplicons (running as adoublet), (b) unincorporated primer and (c) cleaved PCR amplicons. Smallamounts of two fragments, produced by background hydrolysis at theribonucleotide residues within the 5'primer, were also visible runningbetween the primer and cleaved amplicons and running parallel with thecleaved amplicons. In all reactions, PCR product and unincorporatedprimer were visible. Reactions containing template DNA that was wildtype at codon 12 (i.e., those that were fully complementary to theDNAzyme) contained cleaved amplicons. Reactions containing template DNAthat was mutated at codon 12 (i.e., those that were mismatched with theDNAzyme) did not contain cleaved amplicons. Only low levels ofbackground hydrolysis products were visible at this position on the gelin these reactions.

The sequence below is an amplicon that is wild type at position 1 ofcodon 12 (underlined) shown in a conformation wherein the DNAzyme (bold)is hybridising in cis(SEQ ID NO.11).

    5' TATAAACTTGTGGTAGTTGGAgcTGGTGGCGTAGGCAAGAGTGC                                                                               C                                   3' TGAACACCATCAACCT GACCACCGTCGACATAGCAGTT                                                      A G                                                                          G   G                                                                        C     C                                                                      A       T                                                                     A       A                                                                      C     G                                                                        A T C                                              

Based on this invention, and using routine methods of primer design,zymogene primers resulting in the production of DNAzymes can be readilydesigned which specifically cleave mutant K-ras sequences.

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7. R. K. Saiki, et al. (1985) Science 230:1350-1354.

8. Walker, G. T., et al. (1992) Strand Displacement Amplification--anisothermal, in vitro DNA amplification technique. Nucleic Acids Res.20:1691.

9. Jonas, V., et al. (1993) Detection and identification ofMycobacterium tuberculosis directly from sputum sediments byamplification of rRNA. Journal of Clinical Microbiology 31:2410-2416.

10. Fahy, E., et al. (1991) Self-sustained sequence replication (3SR):An iso-thermal transcription-based amplification alternative to PCR. PCRMethods Appl 1: 25-33.

11. Nazarenko, I. A., et al. (1997) A closed tube format foramplification and detection of DNA based on energy transfer. NucleicAcids Research 25: 2516-2521.

12. Tyagi, S. and Kramer, F. R. (1996) Molecular Beacons: Probes thatfluoresce on hybridization. Nature Biotechnology 14: 303-308.

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14. Urdea, M. (1993) Synthesis and characterization of branched DNA(bDNA) for direct and quantitative detection o f CMV, HBV, HCV and HIV.Clin. Chem. 39:725-726.

15. Eigen, M. and Rigler, R. (1994) Sorting single molecules:Application to diagnostics and evolutionary biotechnology. PNAS91:5740-5747.

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22. Carmi, N., et al. (1996) In vitro selection of self-cleaving DNAs.Chemistry and Biology 3:1039-1046.

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 11                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 1                                                         - - ggcctgctga aaatgactga ata           - #                  - #                    23                                                                      - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 59                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 2                                                         - - gagaactgca attcgttgta gctagccttt caggacccac gtccacaaaa tg -             #attctga      59                                                                 - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 3                                                         - - cgtccacaaa atgattctga            - #                  - #                      - # 20                                                                  - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 23                                                              <212> TYPE: RNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 4                                                         - - gagaacgcaa uguucaggac cca           - #                  - #                    23                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 31                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 5                                                         - - tcctgaaagg ctagctacaa cgaattgcag t        - #                  - #              31                                                                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 31                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 6                                                         - - ccactcgatt agctgtatcg tcaagccact c        - #                  - #              31                                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Homo sapiens                                                   - - <400> SEQUENCE: 7                                                         - - cctgctgaaa atgactgaat ataaa          - #                  - #                   25                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 50                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 8                                                         - - ccactctcgt tgtagctagc ctattagctg tatcgtcaag ccactcttgc  - #                  50                                                                         - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 9                                                         - - tataaacttg tggtagttgg agct          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 60                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                            - - <400> SEQUENCE: 10                                                        - - acttgtggta gttggatcgt tgtagctagc cctggtggca gctgtatcgt ca -             #aggcactc     60                                                                 - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 98                                                              <212> TYPE: DNA                                                               <213> ORGANISM: synthetic construct                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Combined D - #NA/RNA Molecule:             amplicon                                                                 - - <400> SEQUENCE: 11                                                        - - tataaacttg tggtagttgg agctggtggc gtaggcaaga gtgccttgac ga -             #tacagctg     60                                                                 - - ccaccagggc tagctacaac gatccaacta ccacaagt      - #                      - #     98                                                                   __________________________________________________________________________

What is claimed is:
 1. A method of detecting the presence of a targetnucleic acid sequence in a sample which comprises(a) contacting thesample, under conditions permitting primer-initiated nucleic acidamplification and catalytic nucleic acid activity, with(i) a DNA primersuitable for initiating amplification of the target, and (ii) a DNAzymogene which encodes, but which itself is the anti-sense sequence of,a catalytic nucleic acid molecule, wherein the primer and zymogene aresituated with respect to each other so that, when the target is present,a single amplified nucleic acid molecule is produced which comprises thesequences of both the target and catalytic nucleic acid molecule; and(b) determining the presence of catalytic nucleic acid activity, therebydetermining the presence of the target nucleic acid sequence in thesample.
 2. The method of claim 1, further comprising the step ofquantitatively determining the amount of catalytic nucleic acid activityin the sample resulting from step (a), and comparing the amount ofactivity so determined to a known standard, thereby quantitativelydetermining the amount of the target nucleic acid sequence.
 3. Themethod of claim 1, wherein the primer and zymogene are on separate DNAmolecules, and the primer-initiated nucleic acid amplification isrolling circle amplification.
 4. The method of claim 1, wherein thesample is contacted with two DNA molecules, each molecule comprising aprimer, and at least one molecule comprising the zymogene wherein theprimer is situated 3' of the zymogene.
 5. The method of claim 4, whereinthe single amplified nucleic acid molecule produced in step (a)(ii)further comprises a nucleotide sequence recognized and cleaved in cis bythe zymogene-encoded catalytic nucleic acid co-residing on the amplifiedmolecule.
 6. The method of claim 5, wherein the zymogene-encodedcatalytic nucleic acid is a 10-23 DNAzyme, and wherein the DNA primer ofstep (a)(i) contains at least one purine ribonucleotide residue whichserves as the 5' side of the site recognized and cleaved in cis by the10-23 DNAzyme.
 7. The method of claim 4, wherein at least two of the DNAmolecules comprise both the primer and zymogene.
 8. The method of claim1, wherein the target nucleic acid sequence is a DNA molecule.
 9. Themethod of claim 1, wherein the target nucleic acid sequence is an RNAmolecule, and step (a) further comprises the step of reversetranscribing the target nucleic acid sequence, if present, to DNA priorto contacting the sample with the primer and zymogene.
 10. The method ofclaim 1, wherein the catalytic nucleic acid molecule is a ribozyme. 11.The method of claim 1, wherein the catalytic nucleic acid molecule is aDNAzyme.
 12. The method of claim 1, wherein catalytic nucleic acidactivity comprises the modification of a detectable chemical substrate,which modification is selected from the group consisting ofphosphodiester bond formation and cleavage, nucleic acid ligation andcleavage, porphyrin metallation, and formation of carbon-carbon, esterand amide bonds.
 13. The method of claim 12, wherein the detectablechemical substrate modification is cleavage of a fluorescently labelednucleic acid molecule.
 14. The method of claim 13, wherein thefluorescently labeled nucleic acid molecule is a DNA/RNA chimera. 15.The method of claim 1, wherein the target nucleic acid sequence is froman organism selected from the group consisting of human, bacterium,mycoplasma and virus.
 16. The method of claim 15, wherein the targetnucleic acid sequence is from a human.
 17. The method of claim 16,wherein the presence of the target nucleic acid sequence in the sampleis indicative of a genetic disorder.
 18. The method of claim 1, whereinthe sample is a forensic sample.
 19. A method of simultaneouslydetecting the presence of a plurality of target nucleic acid sequencesin a sample which comprises(a) contacting the sample, under conditionspermitting primer-initiated nucleic acid amplification and catalyticnucleic acid activity, with(i) a plurality of primers wherein for eachtarget being detected, there exists at least one primer suitable forinitiating amplification of that target, and (ii) a plurality ofzymogenes wherein for each target being detected, there exists at leastone zymogene which encodes, but which itself is the anti-sense sequenceof, a catalytic nucleic acid molecule having distinctly measurableactivity, the primer and zymogene being situated with respect to eachother so that, when the corresponding target is present, a singleamplified nucleic acid molecule is produced which comprises thesequences of both the target and corresponding catalytic nucleic acidmolecule; and (b) simultaneously determining the presence of each of thecatalytic nucleic acid activities, thereby determining the presence ofeach of the corresponding target nucleic acid sequences in the sample.20. The method of claim 19, further comprising the step ofquantitatively determining the amount of each catalytic nucleic acidactivity in the sample resulting from step (a), and comparing the amountof each activity so determined to a known standard, therebyquantitatively determining the amount of each target nucleic acidsequence.
 21. The method of claim 1 or 19, wherein the nucleic acidamplification is performed according to a method selected from the groupconsisting of PCR, SDA and TMA.
 22. A kit for use in determining thepresence of a target nucleic acid sequence in a sample, whichcomprises(a) a primer suitable for initiating amplification of thetarget; (b) a zymogene which encodes, but which itself is the anti-sensesequence of, a catalytic nucleic acid sequence, wherein the primer andzymogene are situated with respect to each other so that, when thetarget is present, a single amplified nucleic acid molecule is producedwhich comprises the sequences of both the target and catalytic nucleicacid molecule; and (c) reagents permitting primer-initiated nucleic acidamplification and catalytic nucleic acid activity.
 23. A kit for use indetermining the presence of a plurality of target nucleic acid sequencesin a sample, which comprises(a) a plurality of primers, wherein for eachtarget being detected, there exists at least one primer suitable forinitiating amplification of that target; (b) a plurality of zymogeneswherein for each target being detected, there exists at least onezymogene which encodes, but which itself is the anti-sense sequence of,a catalytic nucleic acid sequence having distinctly measurable activity,wherein the primer and zymogene are situated with respect to each otherso that, when the corresponding target is present, a single amplifiednucleic acid molecule is produced which comprises the sequences of boththe target and corresponding catalytic nucleic acid molecule; and (c)reagents permitting primer-initiated nucleic acid amplification andcatalytic nucleic acid activity.
 24. The kit of claim 22 or 23, whereinthe nucleic acid amplification is performed according to a methodselected from the group consisting of PCR, SDA and TMA.